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Abstract

Developing effective treatments for obesity and related metabolic disease remains a challenge. One logical strategy targets the appetite-regulating actions of gut hormones such as incretins. One of these incretins, glucose-dependent insulinotropic polypeptide (GIP), has garnered much attention as a potential target: however, whether it is beneficial to boost or block the action of GIP to promote weight loss remains an unresolved question. In this issue of the JCI, Kaneko and colleagues show that antagonizing GIP signaling in the CNS enhances the weight-reducing effects of leptin in rodents with diet-induced obesity. The authors posit that an increase in circulating intestinally derived GIP, as a consequence of overnutrition, acts in the brain to impair hypothalamic leptin action, resulting in increased food intake and body weight gain. This research advances the idea that multiple GIP signaling pathways and mechanisms exist in the obese state and offers intriguing insights into the antiobesogenic consequences of antagonizing brain GIP action.

Authors

Nutrient excess, a major driver of obesity, diminishes hypothalamic responses to exogenously administered leptin, a critical hormone of energy balance. Here, we aimed to identify a physiological signal that arises from excess caloric intake and negatively controls hypothalamic leptin action. We found that deficiency of the gastric inhibitory polypeptide receptor (Gipr) for the gut-derived incretin hormone GIP protected against diet-induced neural leptin resistance. Furthermore, a centrally administered antibody that neutralizes GIPR had remarkable antiobesity effects in diet-induced obese mice, including reduced body weight and adiposity, and a decreased hypothalamic level of SOCS3, an inhibitor of leptin actions. In contrast, centrally administered GIP diminished hypothalamic sensitivity to leptin and increased hypothalamic levels of Socs3. Finally, we show that GIP increased the active form of the small GTPase Rap1 in the brain and that its activation was required for the central actions of GIP. Altogether, our results identify GIPR/Rap1 signaling in the brain as a molecular pathway linking overnutrition to the control of neural leptin actions.

The gut-derived hormone glucose-dependent insulinotropic polypeptide, also known as gastric inhibitory polypeptide (GIP), is a well-established incretin hormone (5–8) that directly acts on β cells to stimulate insulin secretion. GIP has also emerged as a critical player in the control of energy balance under conditions of nutrient excess (9). Circulating levels of GIP are elevated during obesity and after consumption of fat or sugar (5–8). Genetic and pharmacological inhibition of GIP and its receptor protects against high-fat diet–induced (HFD-induced) body weight gain (9–14). Furthermore, GWAS have identified GIP receptor (Gipr) variants that correlate with obesity (15, 16). Interestingly, both GIPR agonism and antagonism improve body weight in obese animals and humans (17–21). Thus, it is of particular interest to elucidate GIPR sites of action and mechanisms mediating its effects on obesity.

First, we confirmed Gipr expression in the brain (22) (Supplemental Figure 1, A–C; supplemental material available online with this article; doi:10.1172/JCI126107DS1). To examine the potential role of brain GIPR, we assessed the direct impact of acute inhibition of brain GIPR on obesity by centrally infusing the neutralizing monoclonal antibody Gipg013, which is a highly specific and potent antagonist of GIPR with a fully characterized mode of action (23). Remarkably, central administration (i.c.v.) of Gipg013 significantly reduced the body weight of HFD-induced obese mice (Figure 1A), whereas no effect was observed in mice treated with an isotype control antibody. Food intake (Figure 1B and Supplemental Figure 2A), and fat mass (Figure 1C) were also significantly reduced in Gipg013-treated obese mice. Blood glucose and serum levels of leptin and insulin were decreased in HFD-induced obese mice treated with Gipg013 (Supplemental Figure 2B). The body weight–lowering effect of Gipg013 is probably attributable to reduced food intake, because energy expenditure did not differ between Gipg013- and control IgG-treated obese mice (Supplemental Figure 3). In contrast, in normal chow–fed lean mice, central Gipg013 administration did not reduce body weight, food intake, or fat mass (Figure 1, D–F), indicating that the effects are specific to diet-induced obesity. In agreement with a recent study (21), peripheral administration of Gipg013 did not reduce weight from the baseline but merely prevented weight gain in HFD-induced obese mice (Supplemental Figure 2, C–F). These data collectively indicate a key role of central GIPR signaling in diet-induced obesity. Central administration of Gipg013 into leptin-deficient ob/ob mice, another mouse model of obesity, did not induce any improvement in energy balance (Figure 1, G–I), suggesting that Gipg013 in the brain acts through leptin signaling. These central effects of GIPR antagonism are different from those in GIPR deficiency in ob/ob mice (9) or obese mice treated peripherally with a GIPR antagonistic antibody (21). The differences might be due to distinct sites of actions of GIPR (e.g., the CNS vs. the periphery). In line with this, brain infusion of Gipg013 significantly decreased expression of the leptin signaling inhibitor Socs3 (Figure 1, J and K). Although peripheral GIPR antagonism was reported to potentiate a weight-lowering effect of GLP-1 agonists (21), we did not detect an enhanced effect of central Gipg013 and liraglutide on weight loss (Figure 1, L–N), suggesting that GLP-1 is probably not involved in the process.

Because central inhibition of GIPR resulted in a leptin-dependent antiobesity effect, we investigated the role of GIPR in leptin action in diet-induced obesity by assessing the response of Gipr-deficient mice (Gipr-KO) (9) and WT mice to exogenously administered leptin. Under normocaloric conditions, central injection of leptin resulted in significantly reduced body weight and suppressed food intake in both Gipr-KO and WT mice (Figure 2A). In contrast, under HFD conditions, WT mice did not exhibit these responses to leptin, demonstrating the expected diminished leptin response induced by HFD feeding; Gipr-KO mice, however, retained their sensitivity to leptin (Figure 2B). Since age-, body weight–, and adiposity-matched littermates were used as controls (Figure 2B and Supplemental Figure 4A), the observed effect of Gipr deficiency on leptin sensitivity was independent of the lean phenotype displayed by Gipr-KO mice. Collectively, our data suggest that Gipr is necessary for diminished responses to exogenous leptin in diet-induced obese mice.

To directly test whether activation of GIPR in the brain negatively regulates hypothalamic leptin actions, we performed a stereotaxic injection of GIP into the lateral ventricle of lean C57BL/6J mice and assessed central leptin sensitivity. We found that i.c.v. infusion of GIP blunted the anorectic response to exogenous leptin (Figure 2C) as well as leptin-dependent hypothalamic phosphorylation of STAT3 (p-STAT3), a critical mediator of leptin actions (Figure 2D). Importantly, we did not observe these inhibitory effects of GIP in mice lacking Gipr (Supplemental Figure 4, B and C), demonstrating that GIP acts through its receptor to blunt leptin-dependent effects. Consistently, GIP increased the hypothalamic levels of Socs3 (Figure 2F). In addition, GIP pretreatment completely blunted leptin-induced neural activation of pro-opiomelanocortin (POMC) neurons, which are known to mediate leptin-induced anorectic responses, whereas leptin depolarized neurons expressing both POMC and the leptin receptor in control slices (Figure 2E and Supplemental Figure 5). Altogether, these data suggest that GIP drives neuronal leptin resistance.

Since endogenous GIP is produced in K cells in the upper gut and GIP levels are reported to be elevated in diet-induced obesity, reaching 20–100 pM (9, 14, 24, 25), we next determined whether increasing the peripheral levels of GIP inhibits neural leptin actions. We administered GIP through i.p. infusions into lean C57BL/6J mice for 3 days and assessed central leptin sensitivity. Peripheral injection of GIP, at a dose to achieve physiological levels similar to those observed in obese animals (Supplemental Figure 6A), markedly blunted anorectic responses to exogenously administered leptin (Figure 2G). Insulin, leptin, and glucose levels were not significantly altered after 3 days of GIP infusion (Supplemental Figure 6, B and C). Given the growing evidence that peripherally injected GIP can reach the brain (refs. 26–28 and Supplemental Figure 7), these data demonstrate that central effects of leptin are partially blunted by peripheral administration of GIP.

Next, we sought to identify an intracellular mediator of GIP action in the brain in ex vivo brain slices. Since GIPR couples to cAMP-related signaling (5–8), we examined the involvement of protein kinase A (PKA) and EPAC, two key downstream components of the cAMP pathway. As previously shown (29), leptin robustly induced hypothalamic p-STAT3 levels in brain slices (Figure 3, A and B, and Supplemental Figure 8A). In contrast, leptin-induced hypothalamic p-STAT3 levels were blunted in the slices pretreated with a native GIP peptide in a dose- and time-dependent manner (Figure 3, A and B). An inactive GIP peptide (GIP3–42) failed to show an inhibitory effect (Supplemental Figure 8C). GIP also increased SOCS3 protein levels ex vivo (Supplemental Figure 8B). We found that the inhibitory effect of GIP was completely blocked with either ESI-05, an EPAC2-specific inhibitor (Figure 3C), or ESI-09, a specific inhibitor for both EPAC1 and EPAC2 (data not shown), but the inhibitory effect of GIP was not affected by the PKA inhibitor PKI14–22 (Figure 3D) or H89 (Supplemental Figure 8D), suggesting that the process is EPAC mediated. Consistently, in ex vivo brain slices, we further observed GIP increases in the amount of the active GTP-bound form of the small GTPase Rap1, which is the direct target of EPAC (Supplemental Figure 8E) or after i.c.v. injection of GIP into lean mice (Figure 3E). In contrast, Gipg013 treatment resulted in a decrease in active Rap1 (Figure 3E). Because neural Rap1 was previously shown to sufficiently drive leptin resistance and be causally related to HFD-induced obesity (4), we reasoned that Rap1 could be a mediator of GIP signaling in the brain. To conclusively test this, we centrally injected GIP into mice with Rap1 deficiency in the forebrain, including multiple hypothalamic nuclei (Rap1ΔCNS) (4, 30), or into control mice. Remarkably, we found that Rap1ΔCNS mice were protected from GIP-mediated leptin resistance and hypothalamic induction of SOCS3 expression, whereas their littermate controls clearly developed GIP-dependent leptin resistance (Figure 3, F and G). Thus, these data indicate that GIP and its receptor are necessary and sufficient for Rap1 activation in the brain and, moreover, that Rap1 activation is required to elicit GIP-induced leptin resistance.

MF conceived the study. KK, YF, HYL, ELC, KWW, and MF designed the experiments. KK, YF, HYL, ELC, YG, TY, KS, PX, SSC, JN, VH, and MHC performed the experiments. PR contributed reagents and intellectually assisted with studies involving Gipg013. KK, YF, ELC, QM, YG, TY, KS, PX, MHC, YX, KWW, JN, VH, PR, and MF analyzed data and interpreted the results. The majority of the manuscript was written by MF, with some help from KK. All authors approved the final version of the manuscript. The order of the co–first authors was determined by their relative contribution to this study.

We are grateful to the members of the Children’s Nutrition Research Center for valuable suggestions; Firoz Vohra and Marta Fiorotto for comprehensive lab animal monitoring system (CLAMS) analyses; Zainab Mabizari and Amy Ng for technical assistance; and Stephanie Sisley and Qiang Tong for comments on the manuscript. We also thank for Alexei Morozov (Virginia Tech Carilion Research Institute) for providing Rap1-floxed mice. This work was supported by grants from the United States Department of Agriculture (USDA) Current Research Information System (CRIS) (6250-51000-055, to MF); the American Heart Association (AHA) (14BGIA20460080, to MF); the NIH (P30-DK079638 and R01DK104901, to MF); the AHA (15POST22500012, to MF); the Uehara Memorial Foundation (201340214, to KK); the NIH (T32HD071839, to ELC); the AHA (13POST13800000 and 15POST22670017, to PX); the NIH (R01DK100699 and DK119169, to KWW); the China Scholarship Council (201406280111, to TY); the CRIS (6250-51000-059, to MHC); and the NIH (P30-DK079638, to MHC). This project was also supported in part by the Genomic and RNA Profiling Core at Baylor College of Medicine, with funding from a P30 Digestive Disease Center Support Grant (NIDDK-DK56338) and a P30 Cancer Center Support Grant (NCI-CA125123).